FUNCTION OF THE HUMAN DNA-ACTIVATED PROTEIN KINASE

真核细胞有多种监测DNA或DNA的机制 结构，其中一些可能对于维持基因组完整性至关重要 包括检测 DNA 链断裂并激活 G1 细胞的一种 循环检查点。 p53 依赖性 G1 检查点机制的丧失 导致基因组不稳定并增加发育的可能性 肿瘤。 检测 DNA 链断裂并激活细胞周期的酶 检查点仍有待确定，但一种 DNA 信号酶可能 涉及的是DNA-PK。 DNA-PK 是一种中等丰富的核 丝氨酸/苏氨酸蛋白激酶在体外被带有切口的 DNA 激活， 间隙、断裂或单链到双链的转变。 最近的研究 强烈建议 DNA-PK 是位点特异性 V(D)J 所必需的 重组和至少一种修复 DNA 双链的途径 休息。 DNA-PK 磷酸化多种核 DNA 结合蛋白， 包括控制转录、DNA 复制的 p53 蛋白， 重组、修复。 因此，DNA-PK还可能调节其他方面 DNA 代谢的过程，包括细胞周期的进展和 细胞对 DNA 链断裂的反应。 通过以下方法可以轻松测量人体细胞提取物中的 DNA-PK 活性： 高度特异性的基于肽的测定，但 DNA-PK 活性的状态 体内无法监测。 该提案的主要目的是 开发监测组织中 DNA-PK 活性的非放射性方法 培养细胞，从而确定影响细胞生长的环境和因素 调节 DNA-PK 活性。 特别感兴趣的是 DNA-PK 是否 被正常核过程激活（或抑制），包括 转录和 DNA 复制，作为细胞周期状态和 应对各种 DNA 损伤。 我们还将确定 DNA-PK 的特定体外底物（包括 p53）是否 在体内被 DNA-PK 磷酸化。 许多药物用于癌症治疗 是造成 DNA 链断裂的 DNA 损伤诱导剂。 如果DNA-PK 参与细胞对 DNA 损伤的反应或调节细胞 周期进展，然后开发可以调节其活性的药物 可能会导致更好的癌症治疗。

Eukaryotic cells have several mechanisms for monitoring DNA or DNA structures, some of which may be critical for maintaining genome integrity including one that detects DNA strand breaks and activates the G1 cell cycle checkpoints. Loss of the p53-dependent G1 checkpoint mechanism leads to genome instability and an enhanced probability of developing tumors. The enzymes that detect DNA strand breaks and activate cell cycle checkpoints remain to be identified, but one DNA signaling enzyme that may be involved is DNA-PK. DNA-PK is a moderately abundant, nuclear, serine/threonine protein kinase activated in vitro by DNAs with nicks, gaps, breaks, or single-to-double strand transitions. Recent studies strongly suggest that DNA-PK is required for site specific V(D)J recombination and for at least one pathway for repairing DNA double-strand breaks. DNA-PK phosphorylates a variety of nuclear, DNA-binding proteins, including the p53 protein, that control transcription, DNA replication, recombination, and repair. Thus, DNA-PK also may regulate other aspects of DNA metabolism including progression through the cell cycle and the cellular responses to DNA strand breaks. DNA-PK activity is easily measured in extracts of human cells by means of a highly specific peptide based assay, but the status of DNA-PK's activity in vivo cannot be monitored. The major aims of this proposal are to develop non-radioactive methods for monitoring DNA-PK activity in tissue culture cells, and hence, to identify the circumstances and factors that regulate DNA-PK activity. Of specific interest is whether DNA-PK is activated (or inhibited) by normal nuclear processes including transcription and DNA replication, as a function of cell cycle status and in response to various kinds of DNA damage. Also we will determine whether specific in vitro substrates of DNA-PK, including p53, are phosphorylated by DNA-PK in vivo. Many agents used in cancer therapies are DNA-damage-inducing agents that create DNA strand breaks. If DNA-PK is involved in the cellular response to DNA damage or in regulating cell cycle progression, then developing drugs that can modulate its activity may lead to better cancer therapies.